Selection of Agents Modulating Gastrointestinal Pain

ABSTRACT

The present embodiments relate to selection of agents effective in reducing or preventing gastrointestinal pain in a subject. Such an agent is selected and identified if it is capable of reducing spontaneous and/or induced transient receptor potential vanilloid 1 (TRPV1) activation.

TECHNICAL FIELD

The present invention relates primarily to modulation ofgastrointestinal pain and in particular to selection of agents, such asLactic Acid bacteria, capable of modulating gastrointestinal pain andthe use of such agents.

BACKGROUND

Gastrointestinal pain is a symptom of many conditions, diseases anddisorders associated with the gastrointestinal tract. Functionalabdominal pain, refers to recurrent abdominal pain. The vast majority ofpatients with recurrent abdominal pain have “functional” or“non-organic” pain, meaning that the pain is not caused by physicalabnormalities. Various motility disorders are also associated with painand constipation or diarrhea. The term is used to describe a variety ofdisorders in which the gut has not developed properly or lost itsability to coordinate muscular activity due to various causes.

Such disorders may manifest in a variety of ways, and includes but arenot limited to the following:

-   -   Abdominal distention    -   Recurrent obstruction    -   Abdominal colicky pain    -   Constipation    -   Gastroesophageal reflux disease    -   Intractable, recurrent vomiting    -   Diarrhea    -   Irritable bowel syndrome (IBS)    -   Inflammatory bowel disease    -   Fecal incontinence    -   Infantile colic    -   Frequent recurrent abdominal pain (FRAP)    -   Regurgitation    -   Food intolerance

In a broad sense, any significant alteration in the transit of foods andsecretions into the digestive tract may be considered an intestinalmotility disorder and this is type of disorder is often associated withgastrointestinal pain.

Proper coordinated movements of the stomach and intestines are requiredto digest and propel intestinal contents along the digestive tract. Thepatterns of contraction and relaxation necessary for proper motility ofthe gastrointestinal (GI) tract are complex and use the nerves andmuscles within the GI walls. Every day, at any time, many factors caninfluence GI motility, e.g. physical exercise and emotional distress.Newborn infants have to develop the complex system of motility in the GItract. Dysfunctional gastrointestinal motility is often associated withGI pain.

Aging, dementia, stroke, Parkinson disease, spinal cord injuries, rectaltears during birthing, diabetes, surgical complications andneuromuscular disorders, e.g. myasthenia gravis, may cause motilitydisorders that are associated with pain.

Irritable bowel syndrome (IBS), a commonly diagnosed disorder ofintestinal motility and GI pain, has been considered a disease of thecolon for decades, but research on GI motility has demonstrated thatunderlying motility disturbances can occur also in the small bowel. IBScan often be accompanied by GI pain and TRPV1 immunoreactivity has beenshown to be markedly increased in IBS patients (Akbar, Yiangou et al,Gut 2008).

Constipation, often associated with GI pain, is the most commondigestive complaint in the United States but despite its frequency,often remains unrecognized until the patient develops secondarydisorders, such as anorectal disorders or diverticular disease. Asmentioned previously, GI pain is a common symptom of constipation.

Constipation is quite common during pregnancy. The muscle contractionsthat normally move food through the intestines slow down because ofhigher levels of the hormone progesterone and possibly extra iron takenas prenatal vitamin. This is often also accompanied by lower abdominalpain.

Constipation is also associated with increased age and the so called“the aging gut” commonly found especially in people over 70 and inchronic care institutions.

At the other end of the aging spectrum intestinal motility disorders,persistent or excessive crying from infant colic is one of the mostdistressing problems of infancy. It is distressing for the infant, theparents, and the involved healthcare professionals. Colic pain oftenstarts and stops abruptly

Intestinal hypermotility secondary to a presumed autonomic imbalancealso has been proposed as one etiology for colic. Many of the mechanismsthat regulate motor activity are immature in infants. The immaturity ofthese mechanisms may result in increased vulnerability to feedingintolerance. Thus, colic may be a common clinical manifestation in thesubpopulation of infants who have maturational dysfunction in one ormore of the aspects of motility regulation and often leading to GI painfor the infant.

Intestinal motility disorders applies to abnormal intestinalcontractions often associated with GI pain, there are many differentkinds of treatments and recommendations for the different disorders,some which work better than many others.

So there is an overall need and specific problems to solve for variousmotility disorders and pain disorders namely; How to best select agentsto prevent or reduce gastrointestinal pain?

Transient receptor potential vanilloid 1 (TRPV1) is a Ca²⁺ permeantcation channel expressed in e.g. the peripheral nervous system (PNS),the central nervous system (CNS), the respiratory system and thegastrointestinal tract. TRPV1 is activated by physical and chemicalstimuli, e.g. temperature, pH change and capsaicin, and is critical forthe detection of nociceptive and thermal inflammatory pain. In thegastrointestinal tract, TRPV1 immunoreactivity can e.g. be found invisceral sensory afferents and TRPV1 cells transmit e.g. gastric painsensation to the higher centers of the brain. TRPV1 is thought to beinvolved in several gastrointestinal conditions that are associated withpain sensations and TRPV1 immunoreactivity has been shown to be markedlyincreased in e.g. IBS (Akbar, Yiangou et al, Gut 2008). As an example ofthis, patients diagnosed with active inflammatory bowel diseasedemonstrate a greatly increased TRPV1 immunoreactivity in colonic nervefibers (Wang, Miyares and Ahern, 2005 J. Physiol.).

Although TRPV1 is considered to be a potential target for developingdrugs to treat different modalities of pain, the widespread expressionof the receptor may result in adverse events limiting the use ofsystemic TRPV1 antagonists in treating gastrointestinal pain. Inparticular, antagonizing the receptor could potentially lead tocardiovascular complications as a result of decreased vasoactive peptiderelease.

SUMMARY OF THE INVENTION

It is a general objective to find agents suitable for reducing orpreventing gastrointestinal pain.

It is a particular objective to provide a method for selecting agents,preferably bacterial strains and more preferably Lactic Acid Bacteria,effective in reducing or preventing gastrointestinal pain.

These and other objectives are met by embodiments as disclosed herein.

An aspect of the embodiments relates to a method for selecting an agenteffective in reducing or preventing gastrointestinal pain in a subject.The method comprises selecting an agent capable of reducing spontaneousand/or induced transient receptor potential vanilloid 1 (TRPV1)activation.

Another aspect of the embodiments relates to an agent selected by theabove identified method.

A further aspect of the embodiments relates to an agent obtainable bythe above identified selection method for use in reducing or preventinggastrointestinal pain in a subject.

Yet another aspect of the embodiments relates to a compositioncomprising an agent obtainable by the above identified selection methodand at least one additional component selected from a group consistingof a pharmaceutically acceptable carrier, a pharmaceutically acceptablediluent, a pharmaceutically acceptable excipient, a foodstuff, a foodsupplement and another preventive or therapeutic agent.

A related aspect of the embodiments defines an agent as defined above ora composition as defined above for use in reducing or preventinggastrointestinal pain in a subject.

Another related aspect of the embodiments defines use of an agentobtainable by the above identified selection method or as defined aboveor a composition as defined above for the manufacture of a medicament, afood product or a food supplement product for reducing or preventinggastrointestinal pain in a subject.

Yet another aspect of the embodiments relates to a method of reducing orpreventing gastrointestinal pain in a subject. The method comprisesadministering an effective amount of an agent obtainable by the aboveidentified selection method or as defined above or a composition asdefined above to the subject.

The present embodiments provide an efficient technology that can be usedto select or identify agents, in particular bacterial strains, such asLactic Acid bacteria, that can be used to reduce or preventgastrointestinal pain in subjects, preferably human subjects, sufferingfrom a disorder or disease causing or is associated withgastrointestinal pain.

BRIEF DESCRIPTION OF THE FIGURES

The embodiments, together with further objects and advantages thereof,may best be understood by making reference to the following descriptiontaken together with the accompanying figures, in which:

FIG. 1 shows mesenteric multiunit spontaneous firing after addition of1×10⁸ DSM 17938 units (cfu)/ml (A), 1×10⁹ cfu/ml DSM 17938 (B), dilutedDSM 17938 conditioned medium (1:5) (C), 1×10⁹ cfu/ml γ-irradiated DSM17938 (D), and diluted medium alone (1:5) (E) (Wilcoxon tests).

FIG. 2 shows effects of DSM 17938 on spontaneous firing rate of spinalafferents. A) Multi-unit firing rate decreased when 1×10⁹ cfu/ml DSM17938 were added to the lumen (Wilcoxon test). B) Left panel, spinalsingle-unit firing was reduced by DSM 17938 (Wilcoxon test). C) Upperpanels, representative traces of spontaneous multiunit discharge beforeand after adding DSM 17938; lower panels, superimposed waveforms of onesingle unit that occurred at times marked by “o” in upper traces.

FIG. 3 shows that DSM 17938 antagonized the excitatory response ofspinal fibers by adding capsaicin to the serosal superfusate. A)Capsaicin dose-response curve from 113 spinal individual single unitswas plotted () and fitted with a three-parameter logistic equation,EC50=200 nM. Max=238±27%; with additional 116 fibers a dose-responsecurve for capsaicin in the presence of 1×10⁹ cfu/ml DSM 17938 wasadditionally plotted (▪) and fitted with the same logistic equation, forwhich EC₅₀=500 nM and Max=129±17% (P=0.7 and P=0.004 for differences inEC₅₀ and Max respectively, extra sum-of-squares F test). B) Summaryscatter plots with means and SEMs showing how single unit responsesvaried with increasing doses of capsaicin in the absence or presence of1×10⁹ cfu/ml DSM. (n) denotes the proportion of single units for eachgroup.

FIG. 4 shows that DSM 17938 or a TRPV1 antagonist reducedistension-evoked excitatory response in spinal single units. A) Scattergraphs showing that adding 1×10⁹ DSM 17938 to the lumen reduced theincrease in spinal single unit firing rate evoked by raisingintraluminal pressure to 48 hPa. B) Adding 10 μM of the TRPV1 antagonist6-Iodonordihydrocapsaicin to the lumen mimicked the effect of adding DSM17938 (Wilcoxon tests).

FIG. 5 shows that DSM 17938 reduced capsaicin-evoked Ca²⁺ rise in dorsalroot ganglia neuron somata. A) 1 μM capsaicin evoked an increase in DRGneurons Ca²⁺ entrance that was dose-dependently diminished byintraluminal DSM 17938 and unaffected by 1×10⁹ cfu/ml JB-1. B) Summaryplot showing how the ratio (F/F₀) of maximal Ca²⁺ fluorescence (F)evoked by capsaicin to baseline Ca²⁺ fluorescence (F₀) varied with DSM17938 concentration or JB-1. (P values, Bonferroni's multiplecomparisons test).

FIG. 6 shows that 9 day feeding with DSM 17938 reduced gastricdistension evoked bradycardia. A) Summary values for percent decreasesin resting heart rate evoked by 40 and 60 mmHg gastric distension (Pvalues, unpaired t tests). B) Summary plots showing how resting heartrate changed over time in response to 60 mmHg gastric distension.(P=0.01, two-way ANOVA test).

FIG. 7 shows that the DSM 17938 induced decrease of the capsaicinexcitatory actions on spinal afferents was mimicked by DSM 17938conditioned medium. Summary plot showing the firing frequency increaseof single-unit spinal fibers induced by 1 μM capsaicin in controlconditions, with DSM 17938 conditioned medium (1:5) or with 1×10⁹ cfu/mlDSM 17938 (P=0.02, one-way ANOVA test; post-hoc P values, Holm-Sidak'smultiple comparisons test).

DETAILED DESCRIPTION

To facilitate understanding of the invention, a number of terms aredefined below.

“Gastrointestinal pain”, also referred to as GI pain, denotes pain inthe gastrointestinal system of a subject. Such gastrointestinal pain isoften caused by or is associated with, i.e. is a symptom of or anailment component of, various diseases and disorders, typically of thegastrointestinal system. Gastrointestinal pain includes general pain inthe gastrointestinal system, often denoted general gastrointestinal painin the art, pain associated with intestinal motility disorders, painfrom inflammatory bowel diseases and irritable bowel syndrome, gastricpain, general abdominal pain, visceral pain, functional abdominal pain,frequent recurrent abdominal pain and pain in other functionalgastrointestinal disorders.

“Functional abdominal pain” refers to recurrent abdominal pain. The vastmajority of patients with recurrent abdominal pain have “functional” or“non-organic” pain, meaning that the pain is not caused by physicalabnormalities.

“Intestinal motility disorders” is used to describe a variety ofdisorders in which the gut has not developed properly or lost itsability to coordinate muscular activity due to various causes. Suchdisorders may manifest in a variety of ways, and includes but are notlimited to the following:

-   -   Abdominal distention    -   Recurrent obstruction    -   Abdominal colicky pain    -   Constipation    -   Gastroesophageal reflux disease    -   Intractable, recurrent vomiting    -   Diarrhea    -   Irritable bowel syndrome (IBS)    -   Inflammatory bowel disease    -   Fecal incontinence    -   Infantile colic    -   Frequent recurrent abdominal pain (FRAP)    -   Regurgitation    -   Food intolerance

In a broad sense, any significant alteration in the transit of foods andsecretions into the digestive tract may be considered an intestinalmotility disorder and this is often associated with gastrointestinalpain.

“Gastric pain” is a collective term used to describe pain or discomfortin the upper abdomen.

In an embodiment, the cause of the gastric pain is selected from thegroup comprising, such as consisting of, non-ulcer dyspepsia, pepticulcer, gastroesophageal reflux disease and gastritis.

In a particular embodiment, the cause of the gastric pain is non-ulcerdyspepsia and/or gastritis.

As used herein the term “firing frequency” is used to measure thesensory spike trains to the brain.

As used herein the term “intraluminal peak pressure” (PPr) is based onintraluminal pressure recordings, where intraluminal pressure changesare measured at the midpoint of the longitudinal axis of the gutsegment. The pressure signal is analyzed and intraluminal peak pressure(PPr) is identified and measured.

As used herein the term “migrating motor complex frequency” (MMCfrequency) is calculated by counting the number of dark MC bands inspatiotemporal maps.

As used herein the term “migrating motor complex velocity” (MMCvelocity) is measured from the slope(s) of each band in thespatiotemporal map generated by migrating motor complexes.

As used herein the term “agent” is used to mean any substance ormaterial including whole cells; microorganisms; conditioned medium;proteins, peptides, enzymes, and/or molecules derived from such aconditioned medium; proteins, peptides, enzymes and/or moleculessecreted or derived from whole cells or microorganisms; or otherbiological or chemical material that can be used to modulategastrointestinal pain in the gastrointestinal system of a mammal. Anexample of preferred agents are bacterial strains, e.g. probioticbacterial strains, and in particular Lactic Acid bacterial strains.Another example of preferred agent is a conditioned medium frombacterial strains, e.g. probiotic bacterial strains, and in particularLactic Acid bacterial strains.

A conditioned medium, sometimes also referred to as conditioned culturemedium, is a (culture) medium in which cells have been cultured for aperiod of time. The cells cultured in the medium “condition” the mediumby releasing or secreting various components or molecules, such asproteins, peptides, enzymes, cytokines, chemokines, chemicals, etc.

It is beginning to be accepted that intestinal microorganisms signal tothe brain as part of the so called microbiome-gut-brain axis. However,very little is known about the role of the gut microbiome in thedevelopment or function of the nervous system. Presently only little isknown about the quantitative nature of the nervous signal relayed fromgut to the central nervous system.

Single sensory neurons, including those among the vagus fibers,represent continuous physical stimuli as patterned spike trains thatencode the nature and intensity of the stimulus. In addition to this,stimuli may be represented in a population code determined by the numberof active fibres in the bundle. All the information reaching the brainvia primary afferents has to be encoded in the language of neuronalspike trains. Therefore, knowing how the sensory spike trains areaffected by various agents, such as commensals, probiotic strains anddifferent substances, enable us to identify new beneficial gutmicroorganisms and their active molecules by their effects on primaryafferent firing as well as new drugs and other compounds that in variousfashion can intervene in this signaling system, particularly bymodulating TRPV1 activation.

The method of the embodiments herein is employed for selecting an agentfor use in reduction or prevention of gastrointestinal pain byinhibiting signaling through the TRPV1 receptor. The method of theembodiments can thereby be used for evaluating agents that potentiallycould be effective in preventing gastrointestinal pain and/or effectivein reducing, inhibiting or treating gastrointestinal pain. Thus, themethod can be used to identify effective agents capable of modulatinggastrointestinal pain associated with the peripheral (enteric) and/orthe central nervous system.

Thus, an aspect of the embodiments relates to a method for selecting anagent effective in, i.e. for use in, reducing or preventinggastrointestinal pain in a subject. The method comprises selecting anagent capable of reducing spontaneous and/or induced transient receptorpotential vanilloid 1 (TRPV1) activation.

The embodiments are thereby based on using the TRPV1 signaling pathwayas a selection tool in identifying agents that are effective inmodulating, in particular preventing or reducing, such as inhibiting ortreating, gastrointestinal pain in subjects suffering from diseases ordisorders involving or causing such gastrointestinal pain and whichcould be denoted gastrointestinal pain diseases or disorders.

The method of the embodiments is typically performed in vitro or ex vivoas is further disclosed herein. However, the agent is preferably capableof reducing spontaneous and/or induced TRPV1 activation in the subjectwhen administered to the subject.

The method of the embodiments may, thus, be used to find suitable agentsfor reducing or preventing gastrointestinal pain in differentgastrointestinal pain disorders and diseases. Agents are chosen toaffect the TRPV1 activation of the subject in a beneficial way in orderto modulate, i.e. preferably prevent, reduce or treat thegastrointestinal pain.

In an embodiment, the disorder or disease associated withgastrointestinal pain to be prevented or treated with an agent selectedby the method is a gastrointestinal pain disorder or disease.

In an embodiment, the gastrointestinal pain is gastric pain.

In an embodiment, the gastrointestinal pain is visceral pain.

In an embodiment, the gastrointestinal pain may be present in a subjectsuffering from colic.

In an embodiment, the gastrointestinal pain may be present in a subjectsuffering from irritable bowel syndrome (IBS).

In an embodiment, the gastrointestinal pain may be present in a subjectsuffering from constipation. Gastrointestinal pain may be present insubjects suffering from intestinal motility disorders as defined in theforegoing.

Thus, in an embodiment the subject is suffering from an intestinalmotility disorder with associated gastrointestinal pain.

The method of the embodiments is based on the unexpected discovery thatgastrointestinal pain including pain associated with differentconditions and disorders such as motility disorders, and manifestedcentrally or peripherally, is connected to TRPV1 activation, which canbe modulated by previously unknown agents, such as Lactic Acid bacteria.

One kind of gastrointestinal pain is visceral pain that results from theactivation of nociceptors of the abdominal viscera (organs). Visceralstructures are highly sensitive to distension (stretch), ischemia andinflammation, but relatively insensitive to other stimuli that normallyevoke pain. Visceral pain is diffuse, difficult to localize and oftenreferred to a distant, usually superficial, structure. It may beaccompanied by symptoms such as nausea, vomiting, changes in vital signsas well as emotional manifestations. The pain may be described assickening, deep, squeezing, and dull. Distinct structural lesions orbiochemical abnormalities explain this type of pain in only a proportionof patients. These diseases are sometimes grouped under gastrointestinalneuromuscular diseases (GINMD).

Persons can also experience visceral pains, often very intense innature, without any evidence of structural, biochemical orhistolopathologic reason for such symptoms.

A nociceptor is a sensory receptor that responds to potentially damagingstimuli by sending action potentials to specific nociceptive neurons (Mor C) which transmit to the anterolateral tracts of the spinal cord(plus a minor vagal projection) and then to the thalamus, andprosencephalon including the insular and cingulate cortices. Criticalfor pain perception originating in the gut pathology is the activationof pain messages from the gut to the central nervous system viaextrinsic primary afferent fibers that travel in mesenteric afferentnerve bundles. The method can be used to screen agents in order toselect agents with desired properties, e.g. reduction in TRPV1activation, for use in reducing or preventing gastrointestinal pain. Theparameter to be measured in the method, i.e. spontaneous and/or inducedTRPV1 activation, can be monitored and determined in different modelsand systems. Using this information, a profile can be obtained usefulfor defining the detailed and potentially nuanced effects that specificagents have on pain signaling in gastrointestinal pain.

Other parameters that can be measured in the method include general painsignaling, nerve firing activity, e.g. mesenteric nerve bundle analysis,and possibly using different in vivo models of gastrointestinal pain.

In an embodiment, TRPV1 activation is measured in cells expressingTRPV1, such as dorsal root ganglion (DRG) neurons, CaCo2 cells oranother standard human intestinal epithelial cell line. Both spontaneousTRPV1 activation and TRPV1 activation after induction by e.g. capsaicin,pH change or temperature can be measured. Generally, any cell or tissuewhich expresses TRPV1 can be used to measure TRPV1 activation accordingto the embodiments.

Thus, in an embodiment the method comprises contacting a cell expressingTRPV1 with an agent to be tested. The method also comprises measuringspontaneous and/or induced TRPV1 activation in the cell following, i.e.after, contacting the cell with the agent to be tested. The methodfurther comprises comparing the measured spontaneous and/or inducedTRPV1 activation with a control TRPV1 activation. In this embodiment,the method further comprises selecting the agent to be tested as anagent effective in reducing or preventing gastrointestinal pain if themeasured spontaneous and/or induced TRPV1 activation is lower than thecontrol TRPV1 activation.

The control TPRV1 activation can be determined according to variousembodiments. For instance, the TRPV1 activation could be predefined anddetermined from TRPV1 expressing cells with an activation level thatrepresents normal or baseline activation corresponding to e.g.substantially no gastrointestinal pain.

However, a preferred embodiment of determining the control TRPV1activation is to use the TRPV1 expressing cell as an internal control.Hence, in an embodiment the method comprises measuring spontaneousand/or induced TRPV1 activation in the cell prior to contacting the cellwith the agent to be tested. The method also comprises determining thecontrol TRPV1 activation based on the spontaneous and/or induced TRPV1activation measured in the cell prior to contacting the cell with theagent to be tested.

In this approach, spontaneous and/or induced TRPV1 activationmeasurements are therefore preferably performed twice: prior tocontacting the cell with the agent to be tested and following contactingthe cell with the agent to be tested.

Alternatively, the spontaneous and/or induced TRPV1 activationmeasurements could be performed in two parallel experiments: oneexperiment where a cell is contacted with the agent to be tested and onecontrol experiment where a cell is not contacted with the agent. Thecells used in the two experiments are then of the same type, such asboth DRG neurons, CaCo2 cells or another standard human intestinalepithelial cell line. The spontaneous and/or induced TRPV1 activation ismeasured in both experiments and then compared to each other.

The cell to be used in the method can be any cell expressing TRPV1including, but not limited, to ex vivo preparations, cell linesexpressing TRPV1, such as human intestinal epithelial cell linesexpressing TRPV1, and primary cells expressing TRPV1.

The measured spontaneous and/or induced TRPV1 activation and the controlTRPV1 activation can generally be expressed as a respective parametervalue or metric, including a value representing spontaneous and/orinduced TRPV1 activation level.

In an embodiment, primary cells such as e.g. dorsal root ganglion (DRG)neurons are used for analyzing TRPV1 activation.

In another embodiment, cell lines expressing TRPV1 such as CaCo2 oranother standard human intestinal epithelial cell line are used foranalyzing TRPV1 activation.

In other embodiments, commercially available cell lines expressing TRPV1are used for analyzing TRPV1 activation, see e.g. cat: no CT6105 fromChantest.

It is also possible to user reporter gene cells, also denoted reportercells in the art, that can be used to monitor and measure TRPV1expression and/or spontaneous and/or induced TRPV1 activation.

Several different methods can be used to study TRPV1 activation,including but not limited to functional analysis using e.g. calciuminflux induced by e.g. capsaicin and/or its prevention by an agent incells expressing TRPV1 and firing frequencies of mesenteric nervebundles, spontaneous and/or induced by e.g. capsaicin. In anotherembodiment, functional assessment of temperature-gated ion-channelactivity using a real-time PCR machine, as described in Reubish,Emerling et al., BioTechniques 2009, can be used to analyze TRPV1activation. In another embodiment, reporter mice for the TRPV1 channelcan be used to investigate TRPV1 activation. Various in vivo paradigmscan also be used to analyze effects on pain in the invention. Thesemethods include e.g. gastric distention and effects on heart rate (seeExample 3) and colorectal distension models.

Hence, in an embodiment, the method comprises measuring Ca²⁺ influx inthe cell induced by capsaicin or another substance capable of inducingTRPV1 activation, e.g. a capsaicin analogue or other substances capableof activating TRPV1. Also exposing the cell to selected physicalconditions can be used to induce TRPV1 activation, including changing pHor temperature. Thus, exposing the cell to acidic pH, basic pH and/orheat (elevated temperatures, typically above about 42° C.) can induceTRPV1 activation. In this embodiment, the Ca²⁺ influx in the cellexpressing TRPV1 is thereby used as a parameter representing TRPV1activation. A reduction in TRPV1 activation, and in particular areduction in pH-induced, heat-induced and/or capsaicin-induced TRPV1activation, can then be measured as a reduction in Ca²⁺ influx. Inanother embodiment, the method comprises measuring temperature-gatedion-channel activity in the cell. In this embodiment, thetemperature-gated ion-channel activity in the cell expressing TRPV1 isused as a parameter representing TRPV1 activation. The temperature-gatedion-channel activity could be spontaneous or induced, such as induced byincreased temperature. A reduction in TRPV1 activation can then bemeasured as a reduction in temperature-gated ion-channel activity in thecell.

In an embodiment, TRPV1 activation is measured using mesenteric afferentnerve bundle experiments. Thus, another parameter that can be measuredis the spontaneous and/or induced, e.g. by capsaicin, pH change or heat,firing frequencies of mesenteric afferent nerve bundles. This techniquecan be used to determine changes in the excitability of the mesentericnerve fibers induced by different agents to be tested. In an embodiment,a gastrointestinal segment is excised with or without the mesentericarcade containing the nerve bundle supplying the segment made up of bothspinal and vagal fibers for nerve bundle recordings ex vivo (see Example1).

In some embodiments, the regional specificity of the gastrointestinaltract is of importance. Appropriate segments for mesenteric nerveanalysis of the method preferably comprises an appropriate nerve bundleto enable the measurement of mesenteric afferent nerve firing. This canconveniently be provided by having a gastrointestinal segment withattached mesenteric tissue (see Example 1). Thus, this embodiment isconveniently carried out on ex vivo segments from an appropriateexperimental animal, for example on mouse gastrointestinal segments(e.g. mouse colon or jejunum segments). The ability to carry out acomparison of the effect of an agent on the small versus the largeintestine could be advantageous, particularly as, depending on theintestinal motility disorder to be treated and the clinical stage andsymptoms thereof, a treatment which is region-specific, e.g. specificfor either the small or large intestine might be beneficial.

In an embodiment afferent mesenteric nerve spinal traffic is analyzedfor specific and selected parts of the gut. It has been surprising tofind that different agents, such as Lactic Acid bacteria, can influenceor modulate gastrointestinal pain signaling systems in one part, but notin another part of the GI-tract, and via different nerve pathways suchas vagal or for visceral pain through the dorsal root ganglion.

Thus, in an embodiment the method comprises contacting an ex vivogastrointestinal segment with attached mesenteric tissue with an agentto be tested. The method also comprises measuring spontaneous and/orinduced mesenteric afferent firing in the ex vivo gastrointestinalsegment following contacting the ex vivo gastrointestinal segment withthe agent to be tested. The method further comprises comparing thespontaneous and/or induced measured mesenteric afferent firing with acontrol mesenteric afferent firing. In this embodiment, the methodfurther comprises selecting the agent to be tested as an agent effectivein reducing or preventing gastrointestinal pain if the measuredspontaneous and/or induced mesenteric affenerent firing is lower thanthe control mesenteric afferent firing.

The control mesenteric afferent firing can be determined as discussed inthe foreging using the ex vivo gastrointestinal segment as an internalcontrol. In such a case, the method comprises measuring spontaneousand/or induced mesenteric afferent firing in the ex vivogastrointestinal segment prior contacting the ex vivo gastrointestinalsegment with the agent to be tested. The method also comprisesdetermining the control mesenteric afferent firing based on thespontaneous and/or induced mesenteric afferent firing measured in the exvivo gastrointestinal segment prior contacting the ex vivogastrointestinal segment with the agent to be tested.

Alternatively, two parallel experiments can be conducted. In one of theman ex vivo gastrointenstinal segment is contacted with the agent to betested and in the other one, control experiment, an ex vivogastrointenstinal segment is not contacted with the agent. Thespontaneous and/or induced mesenteric afferent firing is measured inboth experiments and compared to each other.

Analyzing one or more of these parameters above will result in a methodof selecting agents effective in reducing or preventing gastrointestinalpain.

In a particular embodiment, the ex vivo gastrointestinal segment isselected from an ex vivo colon or jejunum segment. In such a case, themethod comprises contacting an ex vivo colon or jejunum segment withattached mesenteric tissue with the agent to be tested.

Examples of appropriate methods and apparatus for analyzing spontaneousand/or induced TRPV1 activation are described in the Examples andFigures.

Thus, in preferred methods, the analysis presented will give data onspontaneous and/or induced TRPV1 activation. Analyzing one or several ofthese parameters will result in a preferred method of selecting agentseffective in reducing and/or preventing gastrointestinal pain.

The method of the embodiments can, thus, be used to find agents suitablefor treatment, prevention and/or reduction of gastrointestinal pain, byusing the model herein.

The method presented will provide data on spontaneous and/or inducedTRPV1 activation using different models. Analyzing this parameter willresult in a method of selecting for agents effective in reducing orpreventing gastrointestinal pain.

In an embodiment, the method analyses the effect of an agent on TRPV1activation (spontaneous and/or induced by e.g. capsaicin, pH changeand/or heat and its prevention by an agent) and can thus be used as aread out for gastrointestinal pain signalling, i.e. whether or not anagent is likely to have an effect on gastrointestional pain, e.g.visceral pain. An increase or no significant effect in spontaneous orinduced (e.g. by capsaicin, pH change and/or heat) TRPV1 activity isindicative of an agent which is likely to result in an increase ingastrointestinal pain, or no significant effect on gastrointestinalpain, respectively, whereas a decrease in spontaneous and/or induced(e.g. by capsaicin, pH change and/or heat) TRPV1 activity is indicativeof an agent which will reduce gastrointestinal pain. Preferred agentsare thus those that result in a decrease in spontaneous and/or induced(e.g. by capsaicin) TRPV1 activity.

In another embodiment the method analyses the effect of an agent onTRPV1 activity by analyzing spontaneous and/or induced (e.g. bycapsaicin, pH change and/or heat) mesenteric afferent nerve firing (painsignaling) and can thus be used as a read out for gastrointestinal pain,i.e. whether or not an agent is likely to have an effect ongastrointestinal pain, e.g. visceral pain. An increase or no significanteffect on afferent nerve firing is indicative of an agent which islikely to result in an increase in gastrointestinal pain, or nosignificant effect on gastrointestinal pain, respectively, whereas adecrease in afferent nerve firing is indicative of an agent which willreduce gastrointestinal pain. Preferred agents are thus those thatresult in a decrease in afferent nerve firing, e.g. a decrease in thespontaneous and/or induced firing frequency of afferent nerve bundles.

The agent to be tested is added to the chosen system for TRPV1 activityanalysis in any appropriate manner. In order to analyse the effect ofthe agent on pain signalling, the methods are conveniently carried outin the presence and the absence of the agent. For example, the methodstep is carried out before and after the agent is applied. Thus, in suchmethods the effect of the agent is compared to an appropriate control,for example the results in the presence of the test agent are comparedwith the results in the absence of a test agent, e.g. results withbuffer alone as opposed to buffer plus agent.

The inventors have surprisingly found that certain strains of LacticAcid Bacteria, e.g. DSM 17938, can reduce TRPV1 activation in differentex vivo and in vitro models (see Example 1 and 2). Hence, in a preferredembodiment the agent is a bacterial strain, more preferably a LacticAcid bacteria. Thus, the method of the embodiments can advantageously beused to test various Lactic Acid bacteria in order to identify andselect one or more Lactic Acid bacterial strains that are effective inreducing or preventing gastrointestinal pain in a subject as determinedby the method in terms of being able to reduce spontaneous and/orinduced TRPV1 activation.

Another aspect of the embodiments is an agent selected by the method ofthe embodiments, i.e. obtainable by the selection method.

A preferred agent is a microorganism, more preferably a bacterialstrain, preferably a Lactic Acid bacteria, including parts ormetabolites thereof.

Another preferred agent is a conditioned medium from such amicroorganism.

A related aspect of the embodiments defines an agent obtainable by theselection method of the embodiments for use in reducing or preventinggastrointestinal pain in a subject.

In a particular embodiment, the agent is obtainable by the selectionmethod of the embodiments to be capable of reducing spontaneous and/orinduced RPTV1 activation for use in reducing or preventinggastrointestinal pain in a subject.

In an embodiment, for the reduction or prevention of gastrointestinalpain an agent will be selected which preferably acts to decrease painsignalling as assessed by monitoring the effect of the agent onspontaneous and/or induced TRPV1 activation. Such an agent willpreferably act to reduce or decrease TRPV1 activation in DRG neurons, orother TRPV1-expressing cells or tissue. Preferably the agent will act toreduce the spontaneous and/or induced (e.g. by capsaicin, pH changeand/or heat) TRPV1 activity using different in vitro systems or modelsincluding but not limited to primary cells and cell lines expressing theTRPV1 receptor as previously discussed.

In an embodiment, an agent will be selected which preferably acts toreduce or decrease TRPV1 activation in mesenteric afferent nervebundles. The agent will act to reduce the spontaneous and/or induced(e.g. by capsaicin, pH change and/or heat) firing frequences ofmesenteric afferent nerve bundles.

It is clear from the above that the method of the embodiments can alsobe used to select or identify agents which are not appropriate for thetreatment of gastrointestinal pain, for example agents that do not havea beneficial effect on reducing pain signalling. In particular thoseagents which show no effect on this parameter are unlikely to besuitable for the reduction or prevention of gastrointestinal pain. Inaddition, those agents which have an effect of increasing painsignalling as measured by an increase in spontaneous and/or inducedTRPV1 activity are unlikely to be suitable for the reduction orprevention of gastrointestinal pain.

A further aspect of the embodiments relates to a composition comprisingan agent selected by the method of the embodiments and at least oneadditional component. Thus at least one additional component ispreferably selected from a group consisting of a pharmaceuticallyacceptable carrier, a pharmaceutically acceptable diluent, apharmaceutically acceptable excipient, a foodstuff, a food supplementand another preventive or therapeutic agent.

Thus, an embodiment relates to a composition comprising an agentobtainable by the selection method of the embodiemtns and at least oneadditional compontent selected from a group consisting of apharmaceutically acceptable carrier, a pharmaceutically acceptablediluent, a pharmaceutically acceptable excipient, a foodstuff, a foodsupplement and another preventive or therapeutic agent for use inreducing or preventing gastrointestinal pain in a subject.

The at least one additional component can be administered together withthe agent selected according to the embodiments or can be administeredseparately. In addition, the at least one additional component can beadministered at the same time as the agent selected according to theembodiments or at different time points. Suitable administration regimesand timings can readily be determined by the skilled person depending onthe additional component in question.

In an embodiment, the at least one additional component is anyappropriate nutritional component, e.g. a foodstuff or a foodsupplement.

In an embodiment, the another preventive or therapeutic agent can be anyfurther agent, which is useful in the prevention or reduction, such astreatment, of the gastrointestinal pain in question.

In another embodiment, the another preventive or therapeutic agent is anagent capable of affecting gastrointestinal motility and/or mixing. Theanother preventive or therapeutic agent is then preferably capable ofmodulating (increasing or reducing, depending on the condition to betreated as is known in the art and described below) gastrointestinalmotility and/or mixing.

In a further embodiment, the agent of the embodiments may have dualfunctions, i.e. have function on both gastrointestinal pain andgastrointestinal motility and/or mixing.

The agent selected according to present embodiments with the purpose ofbeing effective in reducing or preventing gastrointestinal pain may alsobe subject to another or additional analysis or selection method withthe purpose of determining whether the agent is additionally effectivein modulating gastrointestinal motility and/or mixing. Such an agent maybe interesting to use for prevention or treatment of e.g. motilitydisorders since it addresses both the gastrointestinal pain and themotility alteration in conjunction. Ways of analyzing motility and/ormixing are known in the art. Parameters to be analyzed include, but arenot limited to, MMC frequency, MMC velocity, intraluminal pressure suchas PPr and other functional models.

In motility analyses, changes in gastrointestinal motility induced by anagent can e.g. be detected as an alteration in motility pattern orcontraction amplitudes. Some agents will have no effect at all. An agentwhich can increase gastrointestinal motility, for example by increasingthe MMC frequency and/or MMC velocity and/or intraluminal pressure suchas PPr will likely be useful to treat disorders associated withgastrointestinal pain in which it would be advantageous to increase thepropulsive motility along the digestive tube such as constipation andcolic.

Alternatively, if for example, the intestinal motility disorder fortreatment is one in which it is desired to increase the transit time ofmaterial through the intestine, e.g. disorders involving rapid passagetransit, such as IBS or diarrhea, then an agent of interest will inaddition to its effects on gastrointestinal pain modulation act todecrease gastrointestinal motility, for example by decreasing MMCfrequency or MMC velocity or intraluminal pressure, e.g. PPr. Preferredagents will decrease at least MMC velocity. Preferred agents willdecrease two or more of these parameters, for example will decrease MMCvelocity and MMC frequency or will decrease MMC frequency andintraluminal pressure (e.g. PPr) or will decrease MMC velocity andintraluminal pressure (e.g. PPr). Most preferred agents will decreaseall of these parameters, for example will decrease MMC frequency, MMCvelocity and intraluminal pressure (e.g. PPr). The motility analysis canbe assessed on an appropriate gastrointestinal segment from the small orlarge intestine, for example a jejunal segment for the small intestineor a colon segment for the large intestine. In some embodiments, the useof large intestine, e.g. colon, segments is preferred.

A further aspect of the embodiments relates to an agent selected by themethod of the embodiments or a composition as defined above for use inreducing or preventing gastrointestinal pain in a subject.

A related aspect of the embodiments defines use of an agent selected bythe method of the embodiments, e.g. obtainable by the selection methodaccording to the embodiments, or a composition as defined above for themanufacture of a medicament, a food product or a food supplement productfor reducing or preventing gastrointestinal pain in a subject.

Another related aspect of the embodiments defines a method of reducingor preventing gastrointestinal pain in a subject. The method comprisesadministering an effective amount of an agent selected by the method ofthe embodiments, e.g. obtainable by the selection method according tothe embodiments, or a composition as defined above to the subject.

In an embodiment of these aspects, the agent is a bacterial strain,preferably a Lactic Acid bacterial strain and more preferably aLactobacillus reuteri strain, such as a bacterial strain capable ofreducing spontaneous and/or induced TRPV1 activation, preferably aLactic Acid bacterial strain capable of reducing spontaneous and/orinduced TRPV1 activation and more preferably a Lactobacillus reuteristrain capable of reducing spontaneous and/or induced TRPV1 activation.

In an embodiment of these aspects, the agent is preferably Lactobacillusreuteri DSM 17938. In other embodiment, the agent is another Lactic Acidbacterial strain, i.e. the agent is a Lactic Acid bacterial strain otherthan Lactobacillus reuteri DSM 17938, preferably a Lactobacillus reuteristrain other than Lactobacillus reuteri DSM 17938.

In an embodiment of these aspects, the agent is a conditioned mediumfrom a bacterial strain, preferably from a Lactic Acid bacterial strainand more preferably from a Lactobacillus reuteri strain, such as aconditioned medium from a bacterial strain capable of reducingspontaneous and/or induced TRPV1 activation, preferably from a LacticAcid bacterial strain capable of reducing spontaneous and/or inducedTRPV1 activation and more preferably from a Lactobacillus reuteri straincapable of reducing spontaneous and/or induced TRPV1 activation.

In an embodiment, the agent is a conditioned medium from Lactic Acidbacterial strains, preferably from Lactobacillus reuteri DSM 17938 or aLactobacillus reuteri strain other than Lactobacillus reuteri DSM 17938.The medium used for such conditioned medium may be any mediumappropriate for culturing Lactic Acid bacterial strains known in theart. In an embodiment, MRS (de Man, Rogosa & Sharpe) medium is used asstarting material for producing conditioned medium from Lactic Acidbacterial strains, preferably from Lactobacillus reuteri DSM 17938. Inan embodiment of the invention, conditioned medium from Lactic Acidbacterial strains, preferably from Lactobacillus reuteri DSM 17938, isfreeze-dried before being introduced in or used as a composition.

In another embodiment, one or several components of conditioned mediumfrom Lactic Acid bacterial strains, preferably from Lactobacillusreuteri DSM 17938, are isolated and administered as purified and/orenriched components to a subject in the form of a suitable composition.Examples of such components include proteins, peptides, enzymes andother molecules preferably secreted from the Lactic Acid bacterialstrain into the medium. Also such components extracted directly from theLactic Acid bacterial strains can be used according to the embodiments.

An appropriate mode of administration and formulation of the agent orcomposition, is chosen depending on the site of disease. A preferredmode of administration is oral, however, equally for some treatmentsintravenous or intramuscular injection will be appropriate.

Appropriate doses of the agent or composition can readily be chosen ordetermined by a skilled person depending on the disorder to be treated,the mode of administration and the formulation concerned.

In one embodiment, the TRPV1 receptor is modulated locally, e.g. byusing perorally administered Lactic Acid bacteria that can modulateTRPV1 activation selectively in the GI tract, in a subject sufferingfrom gastrointestinal pain and thereby minimizing any adverse effects insaid subject. It is believed that this preferred administration route ofthe agent, in particular a Lactic Acid Bacterial strain selected by themethod of the embodiments, will mainly affect TRPV1 activation locally,i.e. within the gastrointestinal system. Thus, agent will then have abeneficial effect in preventing or reducing gastrointestinal pain whileminimizing any undesired TRPV1 modulation outside of thegastrointestinal system.

In the methods and uses of the present embodiments described herein, theterms “increase”, “decrease”, “reduce”, etc., refer to a measurablechange in levels, preferably a significant change in levels, morepreferably a statistically significant change, preferably with aprobability value of <0.05.

Preferred subjects are mammals, more preferably humans.

Where the intestinal motility disorder associated with gastrointestinalpain to be treated is constipation then preferred subjects are elderlypatients or pregnant women. An elderly patient will generally beunderstood to be a patient aged 70 or over.

Where the intestinal motility disorder associated with gastrointestinalpain to be treated is colic, preferably this is infantile colic.

The uses of the agents, preferably Lactic Acid bacterial strains,selected according to the method of the embodiments include thereduction, prevention or alleviation of the relevant disorder orsymptoms of disorder (e.g. can result in the modulation of diseasesymptoms). Such reduction, prevention or alleviation of a disorder orsymptoms thereof can be measured by any appropriate assay.

It is an objective of an embodiment to find agents, such as Lactic AcidBacteria, including parts or metabolites thereof, such as present in orextracted from a conditioned medium, suitable for treatment, reduction,prevention or modulation of gastrointestinal pain in e.g. specificmotility disorders and/or other gastrointestinal paindisorders/diseases, by using the model herein based on the effect of theagent on spontaneous and/or induced TRPV1 activity.

In an embodiment the objective is to select a probiotic bacterialstrain, such as Lactic Acid bacterial strain, which can be effective inpreventing or reducing gastrointestinal pain associated withconstipation in humans, especially elderly subjects or pregnant women.

In an embodiment, the objective is to select an agent, for example aLactic Acid bacterial strain, that can be effective in preventing orreducing gastrointestinal pain associated with infantile colic.

In an embodiment the objective is to select an agent, for example aLactic Acid Bacterial strain, that can be effective in treating,preventing or reducing gastrointestinal pain symptoms of Irritable bowelsyndrome (IBS).

The following are some examples of the embodiments, which are not meantto be limiting of the use of the embodiments herein but to showpractical examples in detail how the invention may be used. Example 1relates to a mesenteric nerve bundle experiment showing that DSM 17938inhibits mesenteric nerve firing frequency. Example 2 shows that DSM17938 blocks capsaicin-induced calcium influx in DRG primary cultures.Example 3 demonstrates that DSM 17938 inhibits heart rate slowing evokedby gastric distention.

EXAMPLES Example 1 Mesenteric Nerve Bundle Experiments ExtracellularRecordings

Adult male Swiss Webster mice (20-30 g) were procured from Charles RiverLaboratories (Wilmington, Mass.). The mice were killed by cervicaldislocation. All ensuing procedures were ex vivo.

Segments of distal jejunum (˜2.5 cm) with attached mesenteric tissuewere removed from freshly killed animals and placed in a Sylgard-coatedPetri dish filled with Krebs buffer (in mM): 118 NaCl, 4.8 KCl, 25NaHCO₃, 1.0 NaH₂PO₄, 1.2 MgSO₄, 11.1 glucose, and 2.5 CaCl₂ bubbled withcarbogen (95% 02-5% CO₂). The oral and anal ends of each segment werecannulated with plastic tubing and emptied. The tissue was pinned to theSylgard, and the mesenteric nerve bundle exposed. The Petri dish wasplaced onto the stage of an inverted microscope and the lumen gravityperfused at 0.5-1 ml/min with oxygenated Krebs or Krebs with additives(Perez-Burgos A., Wang B et al., American journal of physiologyGastrointestinal and liver physiology 2013; 304:G211-20). The serosalcompartment was separately perfused with prewarmed (34° C.) Krebs at 3-5ml/min. The nerve bundle was gently sucked into a glass pipette attachedto a patch-clamp electrode holder (CV-7B; Molecular Devices, Sunnyvale,Calif.), and extracellular nerve recordings made using a Multi-Clamp700B amplifier and Digidata 1440A signal converter (Molecular Devices).Electrical signals were bandpass-filtered at 0.1-2 kHz, sampled at 20kHz, and stored on a personal computer running pClamp 10 software(Molecular Devices). Repeated distensions of segments were made byraising intraluminal pressure above 2 hPa. A constant gravity pressurehead of 48 hPa was applied to the Krebs perfusing the lumen and pressureraised by closing the outflow tube for 1 min to a maximum of 3consecutive distensions. Segments were allowed to rest for 9 min betweendistensions. Constitutive multiunit electrical activity was recorded inthe absence of positive intraluminal pressure.

Vagotomy

Subdiaphragmatic vagotomy was carried out as previously described (vander Kleij H, O'Mahony C et al. American journal of physiologyRegulatory, integrative and comparative physiology 2008; 295:R1131-7).Animals were allowed to recover for 10-14 days before harvesting thejejunum and mesenteric tissue for electrophysiological experiments. Shamvagotomies were performed in 3 animals. Postoperatively, the body weightand general health of the mice were measured daily. We found no evidenceof significant differences in weight gain 1 week postsurgery in eithervagotomized or sham-treated animals (data not shown). All vagotomizedmice were tested for completeness of the procedure by recording aftereach experiment the responses to serosal application of cholecystokinin(CCK). Vagotomy was only deemed to have been effective when CCK did notincrease mesenteric nerve firing rate (Perez-Burgos A., Wang B et al.,American journal of physiology Gastrointestinal and liver physiology2013; 304:G211-20).

Drugs and Bacteria

DSM 17938 were donated by BioGaia AB (Stockholm, Sweden), whereasLactobacillus rhamnosus JB-1 were taken from a stock at the Brian-BodyInstitute at McMaster University (Ontario, Canada). All procedures wereas reported previously (Kunze W A, Mao Y K et al., Journal of cellularand molecular medicine 2009; 13:2261-70, Ma X, Mao Y K et al., Americanjournal of physiology Gastrointestinal and liver physiology 2009;296:G868-75, Wang B, Mao Y K, FASEB journal: official publication of theFederation of American Societies for Experimental Biology 2010;24:4078-88). Bacterial numbers were determined optically, and viabilitychecked after plating on growth medium agar plates. Bacteria from frozenstocks were thawed, centrifuged at 2000 rpm for 15 min, and the pelletsuspended in Krebs and again centrifuged and resuspended. Prior to use,bacteria were diluted to working concentrations with Krebs.Cholecystokinin (25-33) sulfated (CCK) was obtained from AnaSpec(Fremont, Calif.); nicardipine, capsaicin and 6-Iodonordihydrocapsaicinfrom Sigma-Aldrich, and co-conotoxin GVIA (ω-Cg-GVIA) and co-conotoxinMVIIC (ω-Cg-MVIIC) from Alomone Labs (Jerusalem, Israel).6-Iodonordihydrocapsaicin and CCK were dissolved in DMSO; capsaicin wasdissolved in ethanol to make stock solution aliquots. On the day of theexperiment the aliquots were diluted in Krebs to working concentrationswith final DMSO and ethanol concentrations of <0.01% and <0.1%,respectively.

Off-Line Data Analysis

Multi- and single-unit spontaneous firing frequencies were measuredusing Clampfit 10.2 (Molecular Devices) and Origin 8.5 (Northampton,Mass.) software. Multi- and single-unit spike recordings were routinelyused to determine changes in the mesenteric nerve fiber firing ratesinduced by exposing the gut to differing stimuli or pharmacologicalagents (Perez-Burgos A., Wang B et al., American journal of physiologyGastrointestinal and liver physiology 2013; 304:G211-20). The timing ofspikes in the multi-unit recording was determined using the peakdetection module of Clampfit, and average firing frequency wascalculated from interspike intervals. Single-units were extracted fromthe multiunit signal by spike shape matching using the spike shapetemplate detection tool of Clampfit (computerized waveform analysis).After running the template detection algorithm, single-unit spikediscrimination was always checked by visual inspection, and nonmatchingspike events were discarded (<0.2%) (Perez-Burgos A., Wang B et al.,American journal of physiology Gastrointestinal and liver physiology2013; 304:G211-20).

Statistics

Data are expressed as means±SE with N referring to the total number ofthe jejunal segments recorded from, and n referring to the number ofsingle fibers activity extracted from multi-unit recordings. Weextracted a maximum of 6 single-units from each multi-unit recording.The Wilcoxon or unpaired t tests were used for paired or unpaired datacomparisons, respectively; one-way and two-way ANOVA with Bonferroni'spost hoc test were used to compare multiple groups as appropriate.Because large variations in spontaneous activity may occur between onepreparation and another in multi-unit neural activity comparisons werepaired with before and after treatment recordings made where each nervebundle served as its own control to assure significant changes in eachtreatment or drug dose. The percentage of increase in firing abovebaseline frequency vs. capsaicin concentration (in the presence orabsence of bacteria) was plotted and this fitted by a logisticdose-response equation [Y=Bottom+(Top−Bottom/1+10^(LogEC50-X))]. Theparameters describing the logistic fits were compared using an extrasum-of-squares F test. All statistical tests were performed using Prismsoftware 5.0 (GraphPad software, San Diego, Calif.).

Effects of DSM 17938 on Mesenteric Nerve Spontaneous Firing Frequency

Luminal DSM 17938 influenced spontaneous multi-unit discharge of themesenteric nerve (Perez-Burgos A., Wang B et al., American journal ofphysiology Gastrointestinal and liver physiology 2013; 304:G211-20).1×10⁹ cfu/ml intraluminal DSM 17938 caused a decrease in spontaneousmulti-unit firing frequency by 22% from to 36.3±8.4 to 28.2±7.2 Hz (N=7,P=0.02, FIG. 1A); DSM 17938 at 1×10⁸ cfu/ml changed the spontaneousdischarge by 19% from 21.6±5.1 to 17.6±6 Hz, N=6, P=0.09, FIG. 1B). DSM17938 conditioned medium (1:5) also decreased the spontaneous dischargeby 37% from 22.6±4.2 to 14.17±2.4 Hz (N=7, P=0.03, FIG. 1C). However,γ-irradiated killed DSM 17938 or broth alone did not decrease afferentexcitability: from 17.84±5.3 to 18.25±4.1 Hz (N=6, P=0.84), and from20.48±1.6 to 21.49±2.8 Hz (N=6, P=0.10) respectively (FIGS. 1D-E).

Vagotomy and Muscular Paralysis Did not Inhibit the Reduction by DSM17938 of Mesenteric Nerve Spontaneous Firing

We investigated whether the effect of DSM 17938 1×10⁹ cfu/ml on thespontaneous discharge was abrogated by prior vagotomy. To controlpossible direct actions of DSM 17938 on smooth muscle cells, we addedhere and in all subsequent experiments of spontaneous firing the L-typeCa²⁺ channel blocker nicardipine (3 μM) which inhibits musclecontractions. Thus, after vagotomy and adding nicardipine, DSM 17938reduced multi-unit firing frequency by 18% from 16.72±1.9 to 13.77±1.9Hz (N=17, P=0.001, FIG. 2A, 2C). The data from vagotomized animals andparalyzed muscle were then analyzed with respect to single-unit firingrates. DSM 17938 reduced single-unit firing frequency by 19% from0.36±0.05 to 0.29±0.03 Hz (n=30, P=0.02, FIG. 2B, 2C); of these fibers,the majority (20/30) showed a decrease in frequency of 36% from0.42±0.06 to 0.27±0.04 Hz (P<0.0001), but the smaller fraction ofremaining fibers increased their firing rate by 29% from 0.24±0.04 to0.31±0.06 Hz (n=10/30, P=0.006).

DSM 17938 Diminished the Spinal Single-Unit Firing Frequency and theResponse to Capsaicin by Insurmountable Partial Antagonism of TRPV1Receptors

We tested whether DSM 17938 could modify the capsaicin-induced firingfrequency increase on single-unit excitation in tissue taken frompreviously vagotomized mice. Capsaicin applied to the serosalcompartment increased the spontaneous multi- and single-unit firingrate, with onset latencies of ˜60 s and in a dose-dependent manner.Given that the TRPV1 capsaicin sensitive receptors desensitize, andsubsequent applications of the agonist may give rise to a diminishedeffect, we examined responses to a range of capsaicin doses (100 nM-100μM) in individual jejunal segments. We did this with and without 20 minof prior intraluminal application of DSM 17938 1×10⁹ cfu/ml (N=25 foreach curve, 5 segments per each concentration; ˜6 spinal single-unitswere analyzed for each segment). The percentage of increase in firingfrequency versus capsaicin concentration or capsaicin plus DSM 17938concentration were plotted and fitted with a three-parameter logisticequation of the form Y═Bottom+(Top-Bottom)/(1+10^(LogEC50-X)). EC50 forcapsaicin alone was 200 nM compared to 500 nM for capsaicin in thepresence of DSM 17938 (P=0.71). The maximal response (Top) obtained withcapsaicin was 238.4±27.5% vs. 129±17% obtained with capsaicin plus DSM17938 (P=0.004, FIG. 3A). In agreement with previous reports, somespinal fibers were not excited by capsaicin. We examined if capsaicinexcited spinal fibers directly, or whether the excitation depended onintramural synaptic transmission from enteric neurons to intraganglionicspinal endings. We added 1 μM capsaicin on the serosal compartment afterintramural synaptic transmission was blocked by adding 500 nM each ofthe Ca²⁺ blockers ω-Cg-GVIA and ω-Cg-MVIIC. Intramural synaptic blockagedid not diminish the capsaicin-evoked excitation which was 187.5±42.9%(n=17) in the absence of the conotoxins and 219.1±72.6 (n=12) in theirpresence (P=0.947, FIG. 3B). We concluded that DSM 17938 action onspinal afferents does not involve intramural synaptic transmission.

Effects of DSM 17938 Conditioned Medium Containing DSM 17938 BacterialReleased Products Reduced Capsaicin Actions on Spinal Fibers

We tested whether the DSM 17938 bacterial products underlie TRPV1antagonism on mesenteric afferents. The DSM 17938 target in themesenteric afferents are predominantly spinal fibers, given that thepercentage of DSM 17938-induced reduction of spontaneous firingfrequency was very similar with and without previous vagotomy (18% vs.22% respectively); then, we applied 1 μM capsaicin in non-vagotomizedmesenteric fibers which received a previous intraluminal application ofDSM 17938 conditioned medium for 20 min. DSM 17938 conditioned medium(1:5) inhibited the capsaicin-induced firing frequency increase (%) onmesenteric single fibers from 187.5±43% (control group, N=17) to74.89±22% (N=14), and was similar to the percentage of increase inducedby capsaicin on spinal fibers with 1×10⁹ cfu/ml DSM 17938 treatment(80.29±22%, N=17) (P=0.02, one-way ANOVA test; FIG. 7).

DSM 17938 or TRPV1 Antagonism Reduced Distension-Evoked Firing

In the absence of nicardipine to allow muscle contraction, we recordedmulti- and single-unit firing frequency of fibers presumed to benociceptors (Grundy D, Gut 2004; 53 Suppl 2:ii5-8). We tested whetherDSM 17938 could reduce their excitatory response evoked by raisingintraluminal pressure to a nociceptive intensity of 48 hPa (36 mmHg)(Perez-Burgos A., Wang B et al., American journal of physiologyGastrointestinal and liver physiology 2013; 304:G211-20). The firstresponse of these fibers to gut distension is generally higher than thatobtained with a subsequent distension, but remains constant for up to 3further distensions (Perez-Burgos A., Wang B et al., American journal ofphysiology Gastrointestinal and liver physiology 2013; 304:G211-20). Wethus used the 2^(nd) of 3 successive distensions for comparisons.Multi-unit firing was 111.5±16.7 Hz during distension, but after addingDSM 17938 for 20 min, distension increased firing to only 86.5±10.6 Hz(N=5, P=0.31). Spinal single-unit firing frequency was 3.01±0.44 Hzduring distension, but in the presence of DSM 17938 firing was 1.71±0.16during distension (n=28, P=0.008, FIG. 4A). The pre-distension firingfrequency was 0.31±0.05 vs. 0.25±0.07 Hz (n=28, P=0.053, FIG. 4A) forcontrol vs. added DSM 17938. The TRPV1 antagonist6-Iodonordihydrocapsaicin (10 μM) mimicked the effect of DSM 17938 onthe single-unit response by decreasing the response to distension from3.26±0.73 to 2.23±0.50 Hz (n=13, P=0.0002, Wilcoxon test, FIG. 4B in theabsence of nicardipine).

Example 2 DSM 17938 Blocked the Ca²⁺ Rise Induced by Capsaicin in DRGNeuronal Primary Cultures Dorsal Root Ganglion (DRG) Primary Cultures

The spinal column was removed from the body, transferred to a beakercontaining ice-cold Krebs, and bisected longitudinally. DRG were exposedand collected from the thoracolumbar levels. Whole DRG were washed twicewith sterile Leibovitz's L-15 medium (GIBCO, Gaithersburg, Md.), andincubated for 40 min in collagenase type 1 at 1 mg/ml (Sigma-Aldrich;Oakville, ON, Canada) and 0.5 ml trypsin (0.25%, GIBCO) in 20 ml L-15 at37° C. After further addition of 5 ml L-15 containing 10% fetal bovineserum (FBS, GIBCO), the ganglia were centrifuged for 5 min at 1,000 rpm,then washed twice with growth medium (L-15, containing 10% FBS, 1%Penicillin/Streptomycin/Glutamine, 1% HEPES and 1% Na Pyruvate). The DRGwere placed in 2 ml of growth medium and triturated 10 times. Theganglia were then centrifuged for 10 s at 500 rpm and the supernatanttransferred to a sterile tube. They were resuspended in 2 ml of growthmedium, triturated repeatedly until the volume of supernatanttransferred was 10 ml, centrifuged for 5 min at 1,000 rpm, and the finalpellet resuspended in 3 ml of growth medium. Neurons were plated onto 3glass bottomed Petri dishes coated with poly-d-lysine (MatTek, Ashland,Mass.). An additional 1.5 ml of growth medium was added to each dishafter 30 min and the whole incubated for 24 h at 37° C. with carbogen.

Ca²⁺ Imaging

DRG neurons were placed within a plexiglass recording dish and loadedwith the Ca²⁺ indicator Fluo-4-AM (8 μM) diluted in Krebs with 0.1%pluronic acid (in DMSO) at 37° C. for 60 min. The dish was placed intothe recording compartment and superfused with fresh Krebs (˜34° C.) for15 min to allow dye wash out. Cells were observed on an invertedmicroscope (Nikon eclipse TE 2000-S, Melville, N.Y.) and imaged using aRolera-XR camera (Surrey, BC, Canada). Fluorescence intensity inindividual neurons was recorded by Simple PCI 6 software (Compix Inc,Imaging systems, Sewickley, Pa.). Drugs were delivered via amicropipette attached to an electronically controlled pressure pulsepuffer (Picospritzer II; General Valve, Fairfield, N.J.) with the tip<100 μm from the cell. Images, recorded at 0.9 frame/s, were stored on alocal hard drive. Image files were analyzed off-line using Image Jsoftware (NIH, USA, http://imagej.nih.gov/ij). The increase in Fluo-4Ca²⁺ when applying capsaicin was measured as the ratio (F/F₀)fluorescence intensity after capsaicin (F) divided by the intensitybefore capsaicin (F₀). Bacteria and drugs were handled as in Example 1.

Results

The specificity of the effect of DSM 17938 on spinal neurons was furtherinvestigated by testing the bacteria's ability to inhibit the responseof the TRPV1 receptor agonist capsaicin. In these experiments, JB-1 wasalso included. JB-1 (Lactobacillus rhamnosus) has previously been shownto reduce pain and firing discharge of DRG single fibers induced bygastric distension (Duncker et al., The Journal of Nutrition 2011).Since the opening of TRPV1 channels raises the intracellular Ca²⁺concentration we used Ca²⁺ imaging of DRG neuronal primary culture forthese experiments. Puffing 1 μM capsaicin onto DRG neurons evoked anincrease in intracellular Ca²⁺ within ˜30 s (FIG. 5A). Since the TRPV1channel may desensitize with repeated ligand exposure, we only appliedcapsaicin once to each culture dish. We then added either DSM 17938 orJB-1 30 min before applying capsaicin. DSM 17938 at 1×10⁹ cfu/mldecreased the fluorescence rise ratio from 2.36±0.31 (control group,N=14) to 1.25±0.04 F/F₀. But, 1×10⁸ cfu/ml DSM 17938 changed F/F₀ to2.67±0.35 and 1×10^(8.5) cfu/ml DSM 17938 to 2.07±0.27. Adding 1×10⁹cfu/ml of JB-1 had little effect and changed the F/F₀ ratio to 2.48±0.19(N=9), which is similar to the ratio obtained with capsaicin alone (FIG.5B). These results demonstrate that DSM 17938 can block the Ca²⁺ riseinduced by capsaicin in DRG neuronal primary cultures.

Example 3 DSM 17938 Inhibited Heart Rate Slowing Evoked by GastricDistension

A total of 17 rats were assigned to 2 groups. Upon arrival, rats wereallowed to acclimatize for 1 week followed by handling for 1 week (10min/d) to minimize stress effects during experiments. Rats were gavagedeach morning for 9 d with either 0.2 ml (1×10⁹ cfu/ml) live DSM 17938 inKrebs or only Krebs as control (vehicle). The methods for GD have beenpreviously published (Tougas, Wang, American Journal of Physiology 1999;277:R272-8). In brief, rats were fasted overnight, anesthetized with amixture of ketamine hydrochloride (75 mg/kg body weight) and xylazine(10 mg/kg body weight) intraperitoneally. Supplemental anesthesia wasgiven as necessary. After a mid-line laparotomy, a distension deviceconsisting of a ball-shaped gastric balloon (2-cm i.d.) affixed to aTeflon catheter (20 cm) was inserted into the stomach through a smallincision in the proximal duodenum and connected to a barostat system(Distender, G&J Electronic, Toronto, Canada). The cardiac response wasmeasured while inflating the balloon with air to pressures of 40 and 60mm Hg for 60 s. Ten min of rest was allowed for recovery after everydistension. Only one set of distensions was applied to each rat to avoidpossible compensatory mechanisms and rats were killed after themeasurements before gaining consciousness. Continuous recordings ofheart rate were performed through a surface electrocardiogram consistingof 3 needle electrodes applied to the left and right shoulders and theright hind legs. The signal was amplified and recorded on a personalcomputer using a commercial data acquisition program (Experimenter'sWorkbench, DataWave Technologies, Loveland, Colo.). The heart rate wasmeasured for 60 s before, during, and after each distension for a totalof 180 s. Heart rate (HR) recording before each distension allowedcorrection for possible baseline changes due to variations in levelsanesthesia and ensured any heart rate response could be linked to thedistension. To control the effect of GD over time, the data arepresented as mean change from resting HR (100%=rest) using the mean HRrecorded for a period of 10 s during distension (10, 20, 30, 40, 50, and60 s). Groups were compared using the mean of HR changes (percent ofresting HR) in all rats of the same group during the 60 s of eachdistension (40 and 60 mm Hg). Bacteria and drugs were handled as inExample 1.

Results

The decrease in heart rate evoked by 40 mm Hg was not changed bygavaging DSM 17938 (P=0.121, FIG. 6A) (N=8 and 9 with vehicle and DSM17938 respectively). Inflation with 60 mm Hg decreased heart rate within10 s which persisted for 30 s during distension (FIG. 6B). Gavaging withDSM 17938 for 9 d prior to testing moderated the response to 60 mm Hg(P=0.028, unpaired t-test, FIG. 6A, 6B). Gastric compliance(volume/pressure) did not differ between animals treated with vehicle orDSM 17938, for gastric distension pressures of 40 or 60 mm Hg (data notshown). These results demonstrate an antinociceptive effect by thebacteria.

1-31. (canceled)
 32. A method for selecting an agent for reducinggastrointestinal pain in a subject, said method comprising: selecting anagent that reduces spontaneous and/or induced transient receptorpotential vanilloid 1, TRPV1, activation, wherein said agent is abacterial strain or conditioned medium from the bacterial strain. 33.The method according to claim 32, further comprising: contacting a cellexpressing TRPV1 with the bacterial strain or conditioned medium fromthe bacterial strain; measuring spontaneous and/or induced TRPV1activation in said cell following the contacting; and selecting saidbacterial strain or conditioned medium from the bacterial strain as anagent effective in reducing gastrointestinal pain when said measuredspontaneous and/or induced TPRV1 activation is lower than control TPRV1activation.
 34. The method according to claim 33, wherein measuring saidspontaneous and/or induced TRPV1 activation comprises measuring Ca2+influx in said cell induced by capsaicin, pH change and/or heat.
 35. Themethod according to claim 33, wherein measuring said spontaneous and/orinduced TRPV1 activation comprises measuring temperature-gatedion-channel activity in said cell.
 36. The method according to claim 32,further comprising: contacting an ex vivo gastrointestinal segment withattached mesenteric tissue with the bacterial strain or conditionedmedium from the bacterial strain; measuring spontaneous and/or inducedmesenteric afferent firing in said ex vivo gastrointestinal segmentfollowing the contacting; and selecting said bacterial strain orconditioned medium from the bacterial strain as an agent effective inreducing gastrointestinal pain when said measured spontaneous and/orinduced mesenteric afferent firing is lower than control mesentericafferent firing.
 37. The method according to claim 32, wherein thebacterial strain is a lactic acid bacterial strain and the conditionedmedium is a conditioned medium from a lactic acid bacterial strain. 38.The method according to claim 37, wherein the lactic acid bacterialstrain is a Lactobacillus reuteri strain and the conditioned medium is aconditioned medium from a Lactobacillus reuteri strain.
 39. The methodaccording to claim 32, wherein selecting said bacterial strain orconditioned medium from the bacterial strain comprises selecting abacterial strain or conditioned medium from the bacterial strain thatreduces spontaneous and/or induced TRPV1 activation and modulatesgastrointestinal motility and/or mixing.
 40. A composition comprising: abacterial strain or conditioned medium from the bacterial strain,wherein the bacterial strain or conditioned medium from the bacterialstrain reduces spontaneous and/or induced transient receptor potentialvanilloid 1, TRPV1, activation; and at least one additional componentselected from a group consisting of a pharmaceutically acceptablecarrier, a pharmaceutically acceptable diluent, a pharmaceuticallyacceptable excipient, a foodstuff, a food supplement and anothertherapeutic agent for use in reducing gastrointestinal pain in asubject, and any combination thereof.
 41. The composition according toclaim 40, wherein the composition comprises said another therapeuticagent and said another therapeutic agent modulates gastrointestinalmotility and/or mixing in said subject.
 42. The composition according toclaim 40, wherein the bacterial strain is a lactic acid bacterial strainand the conditioned medium is a conditioned medium from a lactic acidbacterial strain.
 43. The composition according to claim 40, wherein thebacterial strain is Lactobacillus reuteri and the conditioned medium isa conditioned medium from Lactobacillus reuteri.
 44. The compositionaccording to claim 40, wherein the bacterial strain is Lactobacillusreuteri DSM 17938 and the conditioned medium is a conditioned mediumfrom Lactobacillus reuteri DSM
 17938. 45. A method of reducinggastrointestinal pain in a subject in need thereof, said methodcomprising administering an effective amount of the composition of claim40.
 46. The method of claim 45, wherein the bacterial strain is a lacticacid bacterial strain and the conditioned medium is a conditioned mediumfrom a lactic acid bacterial strain.
 47. The method of claim 45, whereinthe bacterial strain is Lactobacillus reuteri and the conditioned mediumis a conditioned medium from Lactobacillus reuteri.
 48. The methodaccording to claim 47, wherein said bacterial strain is Lactobacillusreuteri DSM 17938 and the conditioned medium is a conditioned mediumfrom Lactobacillus reuteri DSM
 17938. 49. The method according to claim45, wherein said subject is suffering from an intestinal motilitydisorder causing said gastrointestinal pain in said subject.
 50. Themethod according to claim 45, wherein said subject is suffering from adisease selected from a group consisting of colic, irritable bowelsyndrome, constipation causing said gastrointestinal pain in saidsubject, and any combination thereof.